The primary goal of this application is to isolate a caspase inhibitory factor induced by 17-beta-estradiol in primary cultures of human neurons. A group of mammalian cysteinyl caspases is activated in a cell-, insult- and species-specific manner during apoptosis of various cell types. In human neurons, caspase-6 is active during serum deprivation-mediated neuronal apoptosis. We have previously shown that caspase-6 activity is lethal to human neurons in culture. Now, we find that 17 -beta-estradiol but not 17-alpha-estradiol, testosterone, or epitestosterone delay caspase-6 mediated neuronal cell death (Zhang et al. 2001). 17-beta-estradiol-treated neuronal extracts directly inhibit recombinant active caspase-6 in an in vitro assay. In contrast, 17-beta-estradiol does not induce CIF nor prevent caspase-mediated cell death in astrocytes. We conclude that 17-beta-estradiol induces a caspase inhibitory factor (CIF) that is preventing neuronal apoptosis. CIF is induced through estrogen receptors via a non-genomic pathway. We show that CIP is a broad spectrum caspase inhibitor between 10 and 14 kDa in size that is fairly resistant to boiling and proteinase K in neuronal extracts. Our results indicate that 17-beta-estradiol induces a novel inhibitor of active caspases and provide an additional mechanism for the neuroprotective action of 17-beta-estradiol. In this proposal, the primary goal is to identify CIF and determine its role in neuronal survival and cell death. In aim #1, we will biochemically isolate and sequence CIF. In aim #2, we will clone CIF cDNA and obtain antibodies. We will then confirm the role of CIF in neuronal survival and against caspases (aim #3), and determine the mode of activation of CIF (aim #4) and inactivation of caspase-6 (aim #5). Finally, we will study the regulation of CIF expression in normal and AD brains (aim #6). Given the strong epidemiological link of estrogen against Alzheimer's disease and its possible prophylactic role in neuroprotection, our results suggest a novel mechanism of action of 17-Beta-estradiol that could be exploited to promote neuroprotection in injury or neurodegenerative diseases.